Cell information obtaining method and cell information obtaining apparatus

ABSTRACT

A cell information obtaining method comprises causing a plurality of fluorescent substances having different fluorescence wavelengths from each other to be bound to a test substance contained in a cell, applying light to the cell to cause fluorescences having different wavelengths and intensities to be generated from the plurality of fluorescent substances, and obtaining a first fluorescence information and a second fluorescence information on the basis of the generated fluorescences.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from prior Japanese Patent ApplicationNo. 2015-215201, filed on Oct. 30, 2015, entitled “CELL INFORMATIONOBTAINING METHOD AND CELL INFORMATION OBTAINING APPARATUS’ and priorJapanese Patent Application No. 2016-145835, filed on Jul. 25, 2016,entitled “CELL INFORMATION OBTAINING METHOD AND CELL INFORMATIONOBTAINING APPARATUS”, the entire content of which is incorporated hereinby reference.

FIELD OF THE INVENTION

The present invention relates to cell information obtaining methods andcell information obtaining apparatuses.

BACKGROUND

Various biological phenomena including cell proliferation and celldifferentiation involve localization of various molecules such as aprotein, mRNA, and microRNA in a cell. Analysis of localization ofvarious molecules in a cell is expected to lead to elucidation of manybiological phenomena, including analysis of molecule functions, analysisof interaction between proteins, analysis of signal transductionpathways, and the like.

International Publication WO 2005/098430 discloses a method foranalyzing localization of molecules in a cell by use of a fluorescencemicroscope and an imaging flow cytometer.

Even when cells are of the same kind derived from the same origin, theindividual cells are diverse. Thus, for example, some sells havespecific molecules localized in a specific site as shown in FIG. 23A,and other cells have specific molecules localized at another site asshown in FIG. 23B. Moreover, for example, in some cases, due to variousfactors, the amount of molecules is not uniform between cells as shownin FIG. 23C. The inventor has found that, when information is obtainedfrom molecules having diverse distributions and amounts in cells, theobtained results are varied. Thus, a technique is desired that allowsaccurate analysis of molecules having diverse distributions and amountsin cells.

SUMMARY OF THE INVENTION

The scope of the present invention is defined solely by the appendedclaims, and is not affected to any degree by the statements within thissummary.

A first mode of the present invention relates to a cell informationobtaining method. The cell information obtaining method according tothis mode includes: causing a plurality of fluorescent substances havingdifferent fluorescence wavelengths from each other to be bound to a testsubstance contained in a cell; applying light to the cell to causefluorescences having different wavelengths and intensities to begenerated from the plurality of fluorescent substances; and obtaining afirst fluorescence information and a second fluorescence information onthe basis of the generated fluorescences.

In the cell information obtaining method according to this mode, a“plurality of fluorescent substances having different fluorescencewavelengths from each other” means that, when light is applied, aplurality of fluorescent substances respectively emit fluorescenceshaving different wavelengths from each other. In order to causefluorescences having different wavelengths and intensities to begenerated from a plurality of fluorescent substances, in a case where,for example, the wavelengths of excitation light are respectivelydifferent for the plurality of fluorescent substances, a plurality oflights having different wavelengths and intensities are applied to acell. The fluorescence information is an image based on fluorescence,for example. “To cause a plurality of fluorescent substances to be boundto a test substance” means that: the plurality of fluorescent substancesmay not necessarily be bound to all the test substance molecules of thesame kind contained in a cell; and it is sufficient that the pluralityof fluorescent substances are specifically bound to at least some of thetest substance molecules of the same kind.

A second mode of the present invention relates to a cell informationobtaining method. The cell information obtaining method according tothis mode includes: bringing a substrate into contact with a testsubstance contained in a cell, to cause a plurality of fluorescentsubstances having different fluorescence wavelengths from each other tobe generated; applying light to the cell to cause fluorescences havingdifferent wavelengths and intensities to be generated from the pluralityof fluorescent substances; and obtaining a first fluorescenceinformation and a second fluorescence information on the basis of thegenerated fluorescences.

A third mode of the present invention relates to a cell informationobtaining method. The cell information obtaining method according tothis mode includes: causing a fluorescent substance to be bound to atest substance contained in a cell; applying light to the cell to causea fluorescence to be generated from the fluorescent substance;obtaining, from the generated fluorescence, a plurality of fluorescenceshaving different wavelengths and intensities; obtaining a firstfluorescence information and a second fluorescence information on thebasis of the obtained fluorescences; and identifying a distributionstate of the test substance in the cell on the basis of the firstfluorescence information and the second fluorescence information.

A fourth mode of the present invention relates to a cell informationobtaining method. The cell information obtaining method according tothis mode includes: bringing a substrate into contact with a testsubstance contained in a cell, to cause a fluorescent substance to begenerated; applying light to the cell to cause a fluorescence to begenerated from the fluorescent substance; obtaining a plurality offluorescences having different wavelengths and intensities from thegenerated fluorescence; obtaining a first fluorescence information and asecond fluorescence information on the basis of the obtainedfluorescences; and identifying a distribution state of the testsubstance in the cell on the basis of the first fluorescence informationand the second fluorescence information.

A fifth mode of the present invention relates to a cell informationobtaining apparatus. The cell information obtaining apparatus accordingto this mode includes: a light application unit configured to applylight to a cell containing a test substance to which a plurality offluorescent substances having different fluorescence wavelengths fromeach other are bound, thereby to cause fluorescences having differentwavelengths and intensities to be generated from the plurality offluorescent substances; a light receiver configured to receive thefluorescences generated from the plurality of fluorescent substances;and an obtaining section configured to obtain a first fluorescenceinformation and a second fluorescence information on the basis of thefluorescences having different intensities.

A sixth mode of the present invention relates to a cell informationobtaining apparatus. The cell information obtaining apparatus accordingto this mode includes: a light application unit configured to applylight to a cell containing a test substance to which a fluorescentsubstance is bound, thereby to cause a fluorescence to be generated fromthe fluorescent substance; a light receiver configured to receive aplurality of fluorescences having different wavelengths and intensitiesgenerated from the fluorescent substance; an obtaining sectionconfigured to obtain a first fluorescence information and a secondfluorescence information on the basis of the received plurality offluorescences; and an analysis section configured to identify adistribution state of the test substance in the cell on the basis of thefirst fluorescence information and the second fluorescence information.

A seventh mode of the present invention relates to a cell informationobtaining apparatus. The cell information obtaining apparatus accordingto this mode includes: a light application unit configured to applylight to a cell containing a test substance, which has been brought intocontact with a substrate to cause a fluorescent substance to begenerated, thereby to cause a fluorescence to be generated from thefluorescent substance; a light receiver configured to receive aplurality of fluorescences having different wavelengths and intensitiesgenerated from the fluorescent substance; an obtaining sectionconfigured to obtain a first fluorescence information and a secondfluorescence information on the basis of the received plurality offluorescences; and an analysis section configured to identify adistribution state of the test substance in the cell on the basis of thefirst fluorescence information and the second fluorescence information.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart showing a cell information obtaining methodaccording to Embodiment 1;

FIG. 2 shows the outline of how to obtain fluorescence according toEmbodiment 1;

FIG. 3 is a conceptual diagram showing images obtained on the basis ofhigh intensity fluorescence and low intensity fluorescence according toEmbodiment 1;

FIG. 4A shows images obtained in an examination according to Embodiment1;

FIG. 4B shows numerical values obtained in the examination according toEmbodiment 1;

FIG. 5 is a block diagram showing a configuration of an apparatusaccording to Embodiment 1;

FIG. 6 shows a configuration of an optical detection unit according toEmbodiment 1;

FIG. 7 shows a configuration of an optical detection unit according to amodification of Embodiment 1;

FIG. 8 is a flow chart showing a process performed by a cell informationobtaining apparatus according to Embodiment 1;

FIG. 9 shows a screen to be displayed on a display unit according toEmbodiment 1;

FIG. 10 is a conceptual diagram showing graphs obtained on the basis ofhigh intensity fluorescence and low intensity fluorescence according toa modification of Embodiment 1;

FIG. 11 shows the outline of how to obtain fluorescence according toEmbodiment 2;

FIG. 12 shows the outline of how to obtain fluorescence according toEmbodiment 3;

FIG. 13 is a diagram for explaining the transmission wavelength band ofa filter member and wavelength bands of fluorescence to be obtainedaccording to Embodiment 3;

FIGS. 14A and 14B show images obtained in an examination according toEmbodiment 3;

FIG. 15 shows a configuration of an optical detection unit according toEmbodiment 3;

FIG. 16 shows the outline of how to obtain fluorescence according toEmbodiment 4;

FIG. 17 shows a configuration of an optical detection unit according toEmbodiment 4;

FIG. 18 shows the outline of how to obtain fluorescence according toEmbodiment 5;

FIG. 19 shows a configuration of an optical detection unit according toEmbodiment 5;

FIG. 20 shows the outline of how to obtain fluorescence according toEmbodiment 6;

FIG. 21 shows images obtained in an examination according to Embodiment6;

FIG. 22 shows the outline of how to obtain fluorescence according toEmbodiment 7; and

FIGS. 23A, 23B and 23C are schematic diagrams for explaining the problemto be solved by the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiment 1

Embodiment 1 is realized by a cell information obtaining method inwhich: a plurality of fluorescent substances having differentfluorescence wavelengths from each other are caused to be bound to atest substance contained in a cell; and the localization state of thetest substance is identified on the basis of a plurality offluorescences respectively generated from the fluorescent substances. InEmbodiment 1, the test substance is NF-κB. NF-κB being a transcriptionfactor is considered to be present in the cytoplasm in a state of acomplex with IκB, and to move into the nucleus due to degradation of IκBcaused by various stimuli. In Embodiment 1, NF-κB being the testsubstance is specifically labeled with a fluorescent substance, and onthe basis of the fluorescence from the fluorescent substance, whetherNF-κB is present in the cytoplasm or the nucleus is determined. Itshould be noted that the test substance may be a molecule or a proteinother than NF-κB. For example, the test substance may be a transcriptionfactor other than NF-κB, and may be, for example, STAT (SignalTransducer and Activator of Transcription), NFAT (nuclear factor ofactivated T cells), or HIF (hypoxia-inducible factor). Alternatively,the test substance may be mRNA or microRNA. “A plurality of fluorescentsubstances are caused to be bound to a test substance” means that: theplurality of fluorescent substances may not necessarily be bound to allthe test substance molecules of the same kind contained in a cell; andit is sufficient that the plurality of fluorescent substances arespecifically bound to at least some of the test substance molecules ofthe same kind. Furthermore, the identification of the localization stateis not limited to identification of whether the test substance islocalized in the nucleus or localized in the cytoplasm. For example, inthe case of a protruded shape like a neuron, whether the test substanceis localized at the tip of the protrusion may be identified.

As shown in FIG. 1, the cell information obtaining method includes stepsS1 to S4. In the following, a case will be described in which anoperator executes the cell information obtaining method shown in FIG. 1,by using a flow cytometer capable of taking fluorescence images and aprocessing apparatus capable of analyzing the taken images. Each stepshown in FIG. 1 may be executed as a process performed by a cellinformation obtaining apparatus. The configuration and the process for acase where the cell information obtaining apparatus performs each stepin FIG. 1 will be described with reference to FIG. 5 and thereafter.

In step S1, the operator labels NF-κB contained in cells collected froma subject, with fluorescent substances 11 and 12 having differentfluorescence wavelengths from each other. For example, as shown in FIG.2, the fluorescent substances 11 and 12 are bound to NF-κB contained ina cell, via a primary antibody and a secondary antibody. The fluorescentsubstances 11 and 12 may be bound to NF-κB via a plurality of theprimary antibodies, or may be bound to NF-κB via a part of the antibodyor the entirety of the antibody. In addition, in step S1, the operatorlabels the nucleus contained in the cell with a fluorescent substance 13having a fluorescence wavelength different from those of the fluorescentsubstances 11 and 12.

The fluorescent substances 11, 12, and 13 are each a fluorescent dye.The fluorescent substances 11, 12, and 13 are respectively configured toexcite fluorescences having different wavelength bands from each other,upon being irradiated with lights having wavelengths λ1, λ2, and λ3.That is, the wavelengths of lights for exciting fluorescences from thefluorescent substances 11 to 13 are set to be different from each other.In this manner, a sample is prepared in step S1. It should be notedthat, when the test substance is mRNA or microRNA, such a fluorescentsubstances is bound to the test substance via a nucleic acid probe.

In step S2, the operator drives a flow cytometer. Then, the operatorcauses the sample, which contains the cells labeled with the fluorescentsubstances 11 to 13, to flow in a flow cell. Then, the operator causeslights respectively having wavelengths λ1 to λ3 to be applied to eachcell flowing in the flow cell, thereby causing fluorescences to begenerated from the fluorescent substances 11 to 13.

As shown in FIG. 2, when laser lights respectively having wavelengths λ1to λ3 are applied to the cell, fluorescences having different wavelengthbands are generated from the fluorescent substances 11 to 13,respectively. A filter member 21 allows fluorescence having a wavelengthband B1 and having been generated from the fluorescent substance 11 topass therethrough, and blocks light other than light having thewavelength band B1. The fluorescence having the wavelength band B1 andhaving been generated from the fluorescent substance 11 is separated bythe filter member 21. A filter member 22 allows fluorescence having awavelength band B2 and having been generated from the fluorescentsubstance 12 to pass therethrough, and blocks light other than lighthaving the wavelength band B2. The fluorescence having the wavelengthband B2 and having been generated from the fluorescent substance 12 isseparated by the filter member 22. A filter member 23 allowsfluorescence having a wavelength band B3 and having been generated fromthe fluorescent substance 13 to pass therethrough, and blocks lightother than light having the wavelength band B3. The fluorescence havingthe wavelength band B3 and having been generated from the fluorescentsubstance 13 is separated by the filter member 23.

Here, the laser light having the wavelength λ1 is applied to the cell ata high power, and the laser light having the wavelength λ2 is applied tothe cell at a low power. Since the laser light having the wavelength λ1is applied to the cell at a high power, the fluorescence having thewavelength band B1 and having passed through the filter member 21 has ahigh intensity. Since the laser light having the wavelength λ2 isapplied to the cell at a low power, the fluorescence having thewavelength band B2 and having passed through the filter member 22 has alow intensity.

In step S3, the processing apparatus obtains three pieces offluorescence information for each cell, on the basis of fluorescencesrespectively generated from the fluorescent substances 11 to 13. Theflow cytometer includes a configuration in which: fluorescencesrespectively having the wavelength bands B1 to B3 and having beenseparated by the filter members 21 to 23 are caused to form images,respectively, on a light receiver implemented by an image pickup device,thereby obtaining images based on the respective fluorescences. On thebasis of image pickup signals outputted by the light receiver of theflow cytometer, the processing apparatus obtains, as the fluorescenceinformation, an image based on the high intensity fluorescence havingthe wavelength band B1, an image based on the low intensity fluorescencehaving the wavelength band B2, and an image based on the fluorescencehaving the wavelength band B3.

Images of the fluorescences having the wavelength bands B1 to B3 may beindividually taken by three light receivers, respectively, or may betaken by a single light receiver. When the fluorescences having thewavelength bands B1 to B3 are taken by a single light receiver, theoptical system is configured such that the fluorescences having thewavelength bands B1 to B3 form respective images in different regions onthe light receiving surface of the light receiver.

As shown in FIG. 3, in the case of a cell where NF-κB is localized inthe nucleus, images 31 and 32 are obtained in step S3, for example. Theimage 31 is based on the high intensity fluorescence having thewavelength band B1, and the image 32 is based on the low intensityfluorescence having the wavelength band B2. In each of the images 31 and32, a region 33 where the nucleus is present is set. The region 33 isobtained from an image based on the fluorescence of the wavelength bandB3, i.e., an image based on the fluorescence generated from the nucleus,the image having been generated simultaneously with the images 31 and32.

In the case of a cell where NF-κB is localized in the cytoplasm, images41 and 42 are obtained in step S3, for example. The image 41 is based onthe high intensity fluorescence having the wavelength band B1, and theimage 42 is based on the low intensity fluorescence having thewavelength band B2. Also in each of the images 41 and 42, a region 43where the nucleus is present is set. The region 43 is obtained from animage based on the fluorescence having the wavelength band B3 and havingbeen generated simultaneously with the images 41 and 42.

In the case of a cell having a small expression amount of NF-κB, withreference to the image 31, the intensity of the fluorescence isappropriate, and there is a difference in the fluorescence intensitybetween the nucleus and the cytoplasm. Thus, it is possible to identifythat NF-κB is localized in the region 33 of the nucleus. Meanwhile, withreference to the image 32, the intensity of the fluorescence is too low,it is not possible to identify that NF-κB is localized in the region 33of the nucleus. On the other hand, in the case of a cell having a largeexpression amount of NF-κB, with reference to the image 41, theintensity of the fluorescence is too high, and thus, no difference isobserved in the fluorescence intensity between the nucleus and thecytoplasm. Thus, it is not possible to identify whether NF-κB islocalized in the nucleus region 43 or the cytoplasm region which islarger than the nucleus region 43. Meanwhile, with reference to theimage 42, the intensity of the fluorescence is appropriate, and there isa difference in the intensity between the nucleus and the cytoplasm.Thus, it is possible to identify that NF-κB is localized in thecytoplasm region which is larger than the nucleus region 43.

When NF-κB is localized in the cytoplasm, NF-κB is distributed in thecell in such a manner as to surround the nucleus. That is, when the cellis viewed in the image-taking direction, NF-κB is present also on theside nearer to the viewer relative to the nucleus. Thus, in the image 41based on the high intensity fluorescence, strong fluorescence isgenerated also in the nucleus region due to the presence of NF-κB on theside nearer to the viewer relative to the nucleus. Thus, in the case ofthe image 41, appropriate identification on whether NF-κB is localizedin the nucleus or localized in the cytoplasm is less likely to berealized. In contrast, in the case of the image 42 based on the lowintensity fluorescence, although NF-κB that is present on the sidenearer to the viewer relative to the nucleus causes fluorescence to begenerated also in the nucleus region, the intensity of this fluorescenceis low. Thus, in the case of the image 41, even when NF-κB is localizedin the cytoplasm, the localization state can be appropriatelyidentified.

When NF-κB is localized in the nucleus, although NF-κB is partiallydistributed in the cytoplasm, most of NF-κB is distributed in thenucleus. Thus, in the case of the image 31 based on the high intensityfluorescence, strong fluorescence is generated from NF-κB that isdistributed in the nucleus. Thus, localization of NF-κB in the nucleuscan be appropriately identified. On the other hand, in the case of theimage 32 based on the low intensity fluorescence, since fluorescencefrom NF-κB that is distributed in the nucleus is too weak, appropriateidentification on whether NF-κB is localized in the nucleus or localizedin the cytoplasm is less likely to be realized.

As described above, depending on the amount and the distribution ofNF-κB being a test substance in a cell, the appropriate intensity foridentifying localization of NF-κB is different.

Therefore, the power of the laser light having the wavelength λ1 is setsuch that, in a cell where NF-κB is localized in the nucleus,localization of NF-κB in the nucleus can be appropriately identified asshown in the image 31. The power of the laser light having thewavelength λ2 is set such that, in a cell where NF-κB is localized inthe cytoplasm, localization of NF-κB in the cytoplasm can beappropriately identified as shown in the image 42. Accordingly, in acell as the target of the identification, whether NF-κB is localized inthe nucleus or in the cytoplasm, it becomes possible to identifylocalization of NF-κB by using at least one of the two images.

With reference back to FIG. 1, in step S4, the operator identifies thedistribution state of NF-κB being the test substance by referring to theimage based on the high intensity fluorescence having the wavelengthband B1 and the image based on the low intensity fluorescence having thewavelength band B2. Specifically, from the image based on the highintensity fluorescence having the wavelength band B1 and the image basedon the low intensity fluorescence having the wavelength band B2, theoperator selects an image which allows identification of thelocalization position of NF-κB. Then, on the basis of the selectedimage, the operator identifies whether NF-κB is localized in the nucleusor in the cytoplasm of the cell, i.e., the localization state. Insteadof the localization state, the position at which the NF-κB isdistributed, for example, the distribution range of the NF-κB in thecell, may be identified.

As described above, in Embodiment 1, through the adjustment of the twofluorescences generated from the fluorescent substances 11 and 12labeling NF-κB, either one of the image based on the high intensityfluorescence and the image based on the low intensity fluorescenceallows appropriate judgement on the localization of NF-κB. Accordingly,the operator can accurately analyze NF-κB having diverse distribution inthe cell, on the basis of the two images. Specifically, it is possibleto accurately identify the localization state of NF-κB in the cell,i.e., whether NF-κB is localized in the nucleus or in the cytoplasm.

In Embodiment 1, it is conceivable that: even when NF-κB is localized inthe nucleus, if the amount of NF-κB is too large, the intensity of thefluorescence of the image 31 becomes too high; and thus, the intensityof the fluorescence of the image 32 is suitable. Even in such a case, byusing the image 32 having the suitable fluorescence intensity, thelocalization of NF-κB in the nucleus can be determined. Similarly, it isalso conceivable that: even when NF-κB is localized in the cytoplasm,the intensity of the fluorescence of the image 41 is suitable becausethe amount of NF-κB is small and the intensity of the fluorescence ofthe image 42 is too low. Also in such a case, by using the image 41having the suitable fluorescence intensity, the localization of NF-κB inthe cytoplasm can be identified. In this manner, according to Embodiment1, on the basis of the two images, the localization state of NF-κB inthe cell can be accurately identified irrespective of the amount ofNF-κB.

Meanwhile, vascular endothelial cells detach from the inner wall ofblood vessels and flow into blood. Detachment of vascular endothelialcells is caused not only by inflammatory stimuli but also by pressurechange due to compression or the like. In vascular endothelial cellsdetached due to inflammatory stimuli, NF-κB is likely to be localized inthe nucleus. In vascular endothelial cells detached due to stimuli otherthan inflammatory stimuli, NF-κB is less likely to be localized in thenucleus. According to Embodiment 1, localization of NF-κB can beaccurately identified as described above, and thus, the detachmentcaused by inflammatory stimuli among those causes of detachment can beidentified on the basis of whether NF-κB as the signaling molecule islocalized in the nucleus. Thus, the presence/absence of activation ofvascular endothelial cells can be determined. Accordingly, for example,it is possible to determine whether the detachment of vascularendothelial cells has been caused by compression at the time of bloodcollection, or caused by a factor such as a disease. This providesclinical significance.

An image having an intermediate intensity fluorescence may be obtainedby using a middle-power laser light having a wavelength different fromthe wavelengths λ1 and λ2. That is, it may be configured such that:NF-κB is labeled with three fluorescent substances having differentfluorescence wavelengths from each other; three laser lights are appliedto each cell to cause fluorescences having different intensities to begenerated from the three fluorescent substances, respectively; andimages based on the respective fluorescences are obtained. With thisconfiguration, by using the most appropriate image from among the threeimages having different fluorescence intensities, localization of NF-κBcan be more accurately identified. Four or more levels may be employedfor the intensity level of fluorescence to be generated from the testsubstance. Four or more images based on fluorescences respectivelyhaving four or more intensities may be obtained for each cell.

Further, in step S4, on the basis of the localization state per cellidentified as above, the operator obtains the proportion of cells ineach of which NF-κB is localized in a specific site among the cellscontained in the sample. Specifically, when the number of cells for eachof which NF-κB has been identified as being localized in the nucleus isdefined as N1, and the number of cells for each of which NF-κB has beenidentified as being localized in the cytoplasm is defined as N2, theoperator obtains a nuclear localization percentage and a cytoplasmiclocalization percentage according to the following formula. It should benoted that, in step S4, the operator may obtain a nuclear localizationnumber and a cytoplasmic localization number, instead of the nuclearlocalization percentage and the cytoplasmic localization percentage.

Nuclear localization percentage={N1/(N1+N2)}×100

Cytoplasmic localization percentage={N2/(N1+N2)}×100

In step S2, an image of each fluorescence is obtained by use of the flowcytometer as described above, but not limited thereto, an image of eachfluorescence may be obtained as the fluorescence information by use of amicroscope. That is, by use of the microscope, an image based on highintensity fluorescence generated from the fluorescent substance 11, animage based on low intensity fluorescence generated from the fluorescentsubstance 12, and an image corresponding to the nucleus generated fromthe fluorescent substance 13 may be obtained.

Examination of Embodiment 1

Next, examination of Embodiment 1 performed by the inventor will bedescribed.

1. Preparation

As the cells, human cardiac microvascular endothelial cells (HMVEC-C)(Lonza CatNo.CC-7030, Lot No.0000296500 (P4)) were obtained. As theprimary antibody, NF-κB p65 (D14E12) XP Rabbit mAb (Cell SignalingTechnologies #8242S) was obtained. As the secondary antibody, Goatanti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 647 conjugate(Life technologies A-21245), and Goat anti-Rabbit IgG (H+L) SecondaryAntibody, Alexa Fluor 488 conjugate (Life technologies A-11008) wereobtained. To the secondary antibodies, Alexa Fluor 647 and Alexa Fluor488 were bound, as the fluorescent dyes. As the nucleus staining dye,Cellstain Hoechst 33342 solution (DOjinDO H342) was obtained. Other thanthese, EGM-2MV Medium (Lonza Cat No.CC-3202), EGM-2MV SingleQuots Kit(Lonza Cat No. CC-3202), PBS pH7.4 (GIBCO Cat No.10010-023), BSA(LAMPIRE Cat No. 7500805), PFA (WAKO Cat No.160-16061), and TritonX100(Nacalai Tesque CatNo.35501-15) were obtained.

2. Reagent Preparation

Reagents other than FBS of EGM-2MV SingleQuots Kit were added to 500 mLof EGM-2MV Medium, and 100 mL of the mixture was taken into a sterilizedbottle, to create a serum-free medium. To the remaining amount (400 mL)after the creation of the serum-free medium, 20 mL of FBS of SingleQuotsKit was added, to create a culture medium. Paraformaldehyde wasdissolved in pH12 PBS so as to have a final concentration of 8% w/v, andthen the pH was adjusted to 7.4. 1.5 g of BSA was added to and dissolvedin PBS, and PBS was additionally added thereto to obtain 50 mL, whereby3% BSA/PBS was prepared. 0.5 g of BSA was added to and dissolved in PBS,and PBS was additionally added thereto to obtain 50 mL, whereby 1%BSA/PBS was prepared. TritonX100 was adjusted with PBS so as to have afinal concentration of 0.1% w/v.

3. Procedure

HMVEC-C cells were cultured in the EGM-2MV culture medium in accordancewith a manufacturer-recommended protocol. Cells within six passagesafter the purchase thereof were used in this examination. The shelf lifeof the culture medium after opening was set to three weeks. ForTNF-α-stimulated culture, the culture supernatant of about 70% confluentHMVEC-C cells was removed, and an EGM-2MV culture medium to whichRecombinant Human TNF-alpha had been added so as to have a finalconcentration of 25 ng/mL was added. Then, the resultant mixture wasleft still for 1 hour in a 37° C. CO² incubator. The culture medium wasremoved with an electric pipette, with about 3 mL left, and the cellswere detached with a scraper. 8% PFA/PBS was added by an amountequivalent to the collected suspension, and the resultant mixture wasallowed to react at room temperature for 15 minutes. At roomtemperature, centrifugal separation was performed at 1000 rpm for 3minutes. The cell pellet was washed with 1 mL of PBS twice. Thesupernatant was removed, and 1 mL of 0.1% Triton X-100/PBS was added.Then, the resultant mixture was allowed to react at room temperature for15 minutes. At room temperature, centrifugal separation was performed at1000 rpm for 3 minutes. The cells were washed with 1 mL of 1% BSA/PBStwice. The supernatant was removed and 1 mL of 3% BSA/PBS was added.Then, the resultant mixture was left still for 30 minutes at roomtemperature. 400 μL of the primary antibody diluted at a ratio of 1/1600in 3% BSA/PBS was added. The resultant mixture was allowed to react atroom temperature for 1 hour. At room temperature, centrifugal separationwas performed at 1000 rpm for 3 minutes. The cells were washed with 1 mLof 1% BSA/PBS. 400 μL of the secondary antibody diluted at a ratio of1/1000 in 3% BSA/PBS was added. The resultant mixture was allowed toreact at room temperature for 30 minutes. The cells were washed with 1mL of 1% BSA/PBS twice. The supernatant was removed and 50 μL of 1%BSA/PBS was added.

4. Detection by Flow Cytometer

As a flow cytometer that can obtain fluorescence images, ImageStreamXMark II Imaging Flow Cytometer (Merck Millipore) was used. A sampleprepared in accordance with the procedure 3 above was caused to flow inthe flow cell of the flow cytometer. Laser lights respectively havingwavelengths of 488 nm, 647 nm, and 405 nm were applied to the sampleflowing in the flow cell. The laser lights having the wavelengths of 488nm, 647 nm, and 405 nm respectively correspond to laser lights havingthe wavelengths λ1, λ2, and λ3 described above. The emission powers ofthe laser lights having the wavelengths of 488 nm, 647 nm, and 405 nmwere set to 55 mW, 10 mW, and 120 mW, respectively. As a result of theapplication of the laser lights having the wavelengths 488 nm and 647 nmto the two kinds of fluorescent dyes labelling NF-κB, high intensityfluorescence and low intensity fluorescence were generated,respectively. As a result of the application of the laser light havingthe wavelength 405 nm to the nucleus staining dye, fluorescence wasgenerated.

In the flow cytometer above, an image of the fluorescence generated dueto the laser light having the wavelength of 488 nm was taken via afilter member having a transmission wavelength band of 505 nm to 560 nm,whereby a high intensity fluorescence image was obtained. An image ofthe fluorescence generated due to the laser light having the wavelengthof 647 nm was taken via a filter member having a transmission wavelengthband of 642 nm to 740 nm, whereby a low intensity fluorescence image wasobtained. An image of the fluorescence generated due to the laser lighthaving the wavelength of 405 nm was taken via a filter member having atransmission wavelength band of 430 nm to 505 nm, whereby a fluorescenceimage corresponding to the nucleus was obtained. In addition, a laserlight whose wavelength was set between 430 nm to 480 nm was applied tothe sample flowing in the flow cell. An image of light obtained as aresult of this laser light having passed through the cell was taken viathe filter member having a transmission wavelength band of 430 nm to 480nm, whereby a bright field image was obtained. In the flow cytometerabove, light having unnecessary wavelength bands is removed by a filtermember or the like such that light having a target wavelength band isappropriately incident on the light receiver. It should be noted that,in this examination, the bright field image was obtained, but notlimited thereto, a dark field image may be obtained.

With reference to FIG. 4A, images obtained through the detection abovewill be described.

“BRIGHT FIELD” indicates a bright field image of a cell. “HIGH INTENSITYFLUORESCENCE” and “LOW INTENSITY FLUORESCENCE” respectively correspondto an image based on high intensity fluorescence generated from thefluorescent dye labeling NF-κB, and an image based on low intensityfluorescence generated from the fluorescent dye labeling NF-κB.“FLUORESCENCE FROM NUCLEUS” corresponds to an image based onfluorescence generated from the nucleus staining dye that stains thenucleus. “COMBINED” corresponds to an image obtained by combining thefour images on the left. The five images arranged along the horizontaldirection are images obtained from one cell. Images in the columns of“HIGH INTENSITY FLUORESCENCE”, “FLUORESCENCE FROM NUCLEUS”, “LOWINTENSITY FLUORESCENCE”, and “COMBINED” are gray scale expressions ofthe obtained color images, made for convenience. In the images in thecolumns of “HIGH INTENSITY FLUORESCENCE”, “FLUORESCENCE FROM NUCLEUS”,and “LOW INTENSITY FLUORESCENCE”, each white portion indicates that theintensity of the fluorescence is high.

In the case of the cell shown in the upper row, in the image based onthe low intensity fluorescence, the intensity is too low. Thus,localization of NF-κB is difficult to be identified. On the other hand,in the image based on the high intensity fluorescence, the intensity isappropriate. Thus, it is possible to identify that NF-κB is localized inthe nucleus. In the case of the cell shown in the lower row, in theimage based on the high intensity fluorescence, the intensity is toohigh. Thus, localization of NF-κB is difficult to be identified. On theother hand, in the image based on the low intensity fluorescence, theintensity is appropriate. Thus, it is possible to identify that NF-κB islocalized in the cytoplasm.

5. Calculation of Nuclear Localization Percentage

Localization of NF-κB was identified for each cell by viewing theobtained images. This identification was performed in the same manner asdescribed in step S4 above. That is, the nucleus region was set on thebasis of the image of the fluorescence from the nucleus, and the regionother than the nucleus region was set as the cytoplasm region. Then,when it was considered that the fluorescence intensity of the nucleuswas higher than or equal to about twice of the fluorescence intensity ofthe cytoplasm, it was determined that NF-κB was localized in the nucleusin this cell. When it was considered that the fluorescence intensity ofthe nucleus was less than about twice of the fluorescence intensity ofthe cytoplasm, it was determined that NF-κB was localized in thecytoplasm in this cell.

With reference to FIG. 4B, regarding 131 cells recognized by the flowcytometer above, the result of the identification of localization ofNF-κB will be described.

The number of cells for which localization in the nucleus could beidentified was 44, and the number of cells for which localization in thecytoplasm could be identified was 87. The number of cells for whichlocalization was not identified was 0. The nuclear localizationpercentage at this time was: 44/131=34%.

Described below are: Comparative Example 1 in which localization wasidentified only on the basis of high intensity fluorescence images; andComparative Example 2 in which localization was identified only on thebasis of low intensity fluorescence images. In the case of ComparativeExample 1, the number of cells for which localization in the nucleuscould be identified was 42, and the number of cells for whichlocalization in the cytoplasm could be identified was 10. The number ofcells for which localization could not be identified because of theirtoo high fluorescence intensities was 79. The nuclear localizationpercentage in Comparative Example 1 was 81%. In the case of ComparativeExample 2, the number of cells for which localization in the nucleuscould be identified was 16, and the number of cells for whichlocalization in the cytoplasm could be identified was 84. The number ofcells for which localization could not be identified because of theirtoo low fluorescence intensities was 31. The nuclear localizationpercentage in Comparative Example 2 was 16%.

As described above, this examination shows that, when localization is tobe identified on the basis of two fluorescence images having differentintensities as in Embodiment 1, even with respect to the cells for whichidentification could not be made in Comparative Examples 1 and 2,localization of NF-κB can be identified. In addition, when localizationis identified as in Embodiment 1, the number of cells for whichidentification cannot be made is small. Thus, localization of NF-κB inthe cell can be accurately identified. Therefore, according toEmbodiment 1, while the number of cells for which identification cannotbe made is kept at a low level, localization of NF-κB can be accuratelyidentified. Thus, for example, even when the number of cells collectedfrom a subject is small, it is possible to accurately identifylocalization of NF-κB, while ensuring the number of cells for which theidentification can be made.

Embodiment 1 has shown an example in which the localization state ofNF-κB is identified, as an identification of the distribution state of atest substance. However, when the amount of test substance moleculeschanges, the amount of the molecules may be judged. In such a case, theoperator labels the molecules with the fluorescent substances 11 and 12,and the processing apparatus obtains images on the basis of twofluorescences having different intensities generated from thefluorescent substances 11 and 12. When the amount of the molecules islarge, the operator judges the amount by using a low intensityfluorescence image, and when the amount of the molecules is small, theoperator judges the amount by using a high intensity fluorescence image.Accordingly, the amount of the molecules can be accurately judged.

Apparatus Configuration in Embodiment 1

Description will be given of a configuration of a cell informationobtaining apparatus which takes an image of each cell and whichidentifies localization of a test substance in the cell on the basis ofthe cell information obtaining method according to Embodiment 1.

As shown in FIG. 5, a cell information obtaining apparatus 100 includesa processing unit 110, a sample preparation unit 120, an opticaldetection unit 130, a drive unit 140, a display unit 150, an input unit160, and a storage unit 170.

The processing unit 110 is implemented by a microcomputer, a CPU, andthe like. The storage unit 170 is implemented by a RAM, a ROM, a harddisk, and the like. In the storage unit 170, process programs to beexecuted by the processing unit 110 and various data such as images arestored. The processing unit 110 transmits/receives signals to/fromcomponents of the cell information obtaining apparatus 100, and controlsthe components. The processing unit 110 is provided with functions as anobtaining section 111 and an analysis section 112 by a program stored inthe storage unit 170.

The sample preparation unit 120 prepares a sample by mixing cells andreagents in accordance with step S1 shown in FIG. 1. The samplepreparation may be performed by the operator. In such a case, the samplepreparation unit 120 is omitted from the cell information obtainingapparatus 100. The optical detection unit 130 is a flow cytometer. Theoptical detection unit 130 applies light to each cell contained in asample and takes an image of generated fluorescence. The configurationof the optical detection unit 130 will be described later with referenceto FIG. 6. The drive unit 140 drives light sources 301 to 304 of theoptical detection unit 130 described later.

The display unit 150 is implemented by a display. The display unit 150displays images obtained for each cell, localization of NF-κB identifiedfor each cell, the number of cells in each of which NF-κB is localizedin the nucleus, the number of cells in each of which NF-κB is localizedin the cytoplasm, a nuclear localization percentage, a cytoplasmiclocalization percentage, and the like. The input unit 160 is implementedby a mouse and a keyboard. The operator inputs instructions to the cellinformation obtaining apparatus 100 via the input unit 160.

As shown in FIG. 6, the optical detection unit 130 includes a flow cell200, a light application unit 300, a light condensing unit 400, andlight receivers 501 to 504. The flow cell 200 has a flow pass 210 formedtherein. In the flow pass 210, the sample prepared by the samplepreparation unit 120 is caused to flow. In FIG. 6, XYZ axes orthogonalto one another are shown for convenience.

The light application unit 300 applies light to each cell contained inthe sample flowing in the flow cell 200, thereby to cause fluorescencehaving different intensities to be generated from the fluorescentsubstances 11 and 12 shown in FIG. 2. In addition, the light applicationunit 300 applies light to the cell, thereby to cause fluorescence to begenerated from the fluorescent substance 13 shown in FIG. 2. Further,the light application unit 300 applies light for bright field to thecell. The light application unit 300 includes the light sources 301 to304, condenser lenses 311 to 314, and dichroic mirrors 321 and 322.

The light sources 301 to 304 are each implemented by a semiconductorlaser light source. Lights emitted from the light sources 301 to 304 arelaser lights having the wavelengths λ1 to λ4, respectively. Thewavelengths λ1 to λ4 respectively are 488 nm, 647 nm, 405 nm, and 785nm, for example. The laser lights having the wavelengths λ1 to λ3 arelights for exciting fluorescences from the fluorescent substances 11 to13, respectively, as shown in FIG. 2. The condenser lens 311 to 314condense lights emitted from the light sources 301 to 304, respectively.The dichroic mirror 321 allows light having the wavelength λ1 to passtherethrough, and reflects light having the wavelength λ2. The dichroicmirror 322 allows lights having the wavelengths λ1 and λ2 to passtherethrough, and reflects light having the wavelength λ3.

Thus, the light application unit 300 applies, in a superposed manner,the lights having the wavelengths λ1 to λ3 emitted from the lightsources 301 to 303, to each cell contained in the sample flowing in theflow pass 210. In addition, the light application unit 300 applies lighthaving the wavelength λ4 to the position, in the flow pass 210, at whichthe lights having the wavelengths λ1 to λ3 are applied. When the lightshaving the wavelengths λ1 to λ3 are applied to the sample flowing in theflow cell 200, fluorescences having different wavelength bands aregenerated from the fluorescent substances 11 to 13, respectively, asdescribed with reference to FIG. 2. When light having the wavelength λ4is applied to the sample flowing in the flow cell 200, this light passesthrough the cell. The light having the wavelength λ4 that has passedthrough the cell is used for obtaining a bright field image.

Here, the light source 301 emits light having the wavelength λ1 at ahigh power, and the light source 302 emits light having the wavelengthλ2 at a low power. The emission power of each of the light sources 301and 302 is controlled by the drive unit 140 shown in FIG. 5.Accordingly, as described with reference to FIG. 2, the fluorescencegenerated from the fluorescent substance 11 has a high intensity, andthe fluorescence generated from the fluorescent substance 12 has a lowintensity. It should be noted that in Embodiment 1 and Embodiments 2 and3 described later, the emission power of each of the light sources 301and 302 may not be adjusted. If a fluorescence label is selected thathas the same emission power but that causes a difference in obtainedfluorescence intensities, fluorescence generated from the fluorescentsubstance 11 will have a high intensity, and fluorescence generated fromthe fluorescent substance 12 will have a low intensity.

The light condensing unit 400 is for condensing fluorescences generatedat the flow cell 200 as a result of application of lights having thewavelengths λ1 to λ3. The light condensing unit 400 causes fluorescencesgenerated from the fluorescent substances 11 to 13, to be condensed ontothe light receivers 501 to 503, respectively. The light condensing unit400 causes light having the wavelength λ4 generated at the flow cell200, to be condensed onto the light receiver 504. The light condensingunit 400 includes a condenser lens 401, filter members 411 to 413 and421 to 424, and condenser lenses 431 to 434.

The condenser lens 401 condenses fluorescence generated from the sampleflowing in the flow cell 200, and light having the wavelength λ4 thathas passed through the sample flowing in the flow cell 200. The filtermembers 411 to 413 are each implemented by a dichroic mirror.

Among the lights condensed by the condenser lens 401, the filter member411 reflects the light having the wavelength band B1 and allows lightother than the light having the wavelength band B1 to pass therethrough.Of the light reflected by the filter member 411, the filter member 421allows only the light having the wavelength band B1 to passtherethrough, and blocks light other than the light having thewavelength band B1. In this manner, the filter members 411 and 421 areeach configured to be able to separate only the fluorescence having thewavelength band B1, among the lights generated at the flow cell 200.Similarly, the filter members 412 and 422 are each configured to be ableto separate only the fluorescence having the wavelength band B2, amongthe lights generated at the flow cell 200. The filter members 413 and423 are each configured to be able to separate only the fluorescencehaving the wavelength band B3, among the lights generated at the flowcell 200. Among the lights that have passed through the filter members411 to 413, the filter member 424 allows the light having the wavelengthλ4 to pass therethrough and blocks light other than the light having thewavelength λ4.

The light receiver 501 receives the light having the wavelength band B1and having been condensed by the condenser lens 431, and outputs, as animage pickup signal, image information based on the received light. Thelight receiver 502 receives the light having the wavelength band B2 andhaving been condensed by the condenser lens 432, and outputs, as animage pickup signal, image information based on the received light. Thelight receiver 503 receives the light having the wavelength band B3 andhaving been condensed by the condenser lens 433, and outputs, as animage pickup signal, image information based on the received light. Thelight receiver 504 receives the light having the wavelength λ4 andhaving been condensed by the condenser lens 434, and outputs, as animage pickup signal, image information based on the received light. Thelight receivers 501 to 504 are each implemented by an image pickupdevice such as a color CCD, for example.

The light condensing unit 400 causes the lights having the wavelengthbands B1 to B3 and the light having the wavelength λ4 to be condensedonto the light receivers 501 to 504, respectively. However, the lightcondensing unit 400 may cause those lights to form images on a singlelight receiver. In such a case, the optical detection unit 130 isconfigured such that the lights having the wavelength bands B1 to B3 andthe light having the wavelength λ4 form images in different regions,respectively, on the light receiving surface of the light receiver.

In the configuration shown in FIG. 6, a plurality of filter members areused in order to separate the lights having the wavelength bands B1 toB3. However, as shown in FIG. 7, the lights generated at the flow cell200 may be separated by a single filter member. As shown in FIG. 7, thelight condensing unit 400 includes condenser lenses 441 to 444, filtermembers 451 to 454, and condenser lenses 461 to 464. Lights having thewavelength bands B1 to B3 are separated by the filter members 451 to453, respectively, and light having the wavelength λ4 is separated bythe filter member 454.

Next, with reference to the flow chart shown in FIG. 8, a case where theprocess of step S4 shown in FIG. 1 is performed by the cell informationobtaining apparatus 100 will be described.

As shown in FIG. 8, in step S11, the processing unit 110 drives thesample preparation unit 120. As in step S1 shown in FIG. 1, theprocessing unit 110 causes the sample preparation unit 120 to labelNF-κB contained in each cell with the fluorescent substances 11 and 12,and to label the nucleus contained in the cell with the fluorescentsubstance 13, thereby preparing a sample. In step S12, the processingunit 110 causes the sample to flow in the flow cell 200. The processingunit 110 causes the drive unit 140 to drive the light sources 301 to304, and then, causes light to be applied to the cell flowing in theflow cell 200. In step S13, the processing unit 110 causes the lightreceivers 501 to 503 to take images of fluorescences having thewavelength bands B1 to B3, respectively. The processing unit 110 causesthe light receiver 504 to take an image of light having the wavelengthλ4. Then, the obtaining section 111 of the processing unit 110 obtainsimages on the basis of image pickup signals outputted by the lightreceivers 501 to 504.

In step S14, the analysis section 112 of the processing unit 110 selectsan image that allows identification of localization, from a highintensity fluorescence image and a low intensity fluorescence image.Specifically, from the high intensity fluorescence image and the lowintensity fluorescence image, the analysis section 112 selects an imagein which the intensity (e.g., the brightness of the entire image) of thefluorescence obtained from the image is within a predetermined range.Accordingly, an image having an extremely high fluorescence intensityand an image having an extremely low fluorescence intensity are excludedfrom images to be used in identification of localization of NF-κB,because such images do not enable the identification as in the case ofthe image 32 and the image 41 in FIG. 3.

In addition, from the high intensity fluorescence image and the lowintensity fluorescence image, the analysis section 112 selects an imagein which the difference between the fluorescence intensity at ananalysis target site of the cell and the fluorescence intensity in theportion other than the analysis target site in the cell is greater thana predetermined threshold. In Embodiment 1, the analysis target site isnucleus. That is, an image is selected in which the difference betweenthe fluorescence intensity inside the nucleus and the fluorescenceintensity in the portion outside the nucleus in the cell is greater thana predetermined threshold. As a result, an image having a smalldifference between the fluorescence intensity inside the nucleus and thefluorescence intensity outside the nucleus is excluded from images to beused in identification of localization of NF-κB, because such an imagedoes not enable the identification as in the case of the image 32 andthe image 41 in FIG. 3.

Next, in step S15, the analysis section 112 identifies localization ofNF-κB in each cell, by using the image selected in step S14. That is,the analysis section 112 calculates the proportion of the localizationamount of NF-κB at the analysis target site in the cell relative to thelocalization amount of NF-κB in the entirety of the cell. For example,with respect to the image selected in step S14, the analysis section 112divides the fluorescence intensity in the nucleus region by thefluorescence intensity in the entire region of the cell. When thedivision result is 2 or greater, the analysis section 112 identifiesthat NF-κB is localized in the nucleus. When the division result issmaller than 2, the analysis section 112 identifies that NF-κB islocalized in the cytoplasm. The criterion value used for determining thedivision result is not limited to 2, and may be another value.

In a case where two images are selected in step S14, then, in step S15,with respect to each of both images, the division result is obtained andlocalization is identified, as described above. In a case where no imageis selected in step S14, then, in step S15, localization of this cell iscategorized as “cannot be identified”.

In step S16, on the basis of the identification results of all the cellsthat have been subjected to the process, the analysis section 112calculates the nuclear localization number, the cytoplasmic localizationnumber, the nuclear localization percentage, and the cytoplasmiclocalization percentage described above. In step S17, the processingunit 110 causes the display unit 150 to display the numerical valuescalculated in step S16, the images obtained for each cell, theidentification result for each cell, and the like. Specifically, theprocessing unit 110 causes the display unit 150 to display a screen 161including the above contents.

As shown in FIG. 9, the screen 161 includes regions 161 a and 161 b. Theregion 161 a shows the nuclear localization number, the cytoplasmiclocalization number, the nuclear localization percentage, and thecytoplasmic localization percentage. The region 161 b shows the imagesand the results of identification of NF-κB localization. In the region161 b, the image used in the identification of localization in step S15is surrounded by a solid line so as to indicate that the image was usedin the localization identification.

The fluorescence information obtained in step S13 may be a waveformsignal that indicates fluorescence intensity changing with time. In sucha case, in the optical detection unit 130, photodetectors such asphotomultipliers are disposed as the light receivers 501 to 503. Threephotodetectors receive the high intensity fluorescence having thewavelength band B1, the low intensity fluorescence having the wavelengthband B2, and the fluorescence having the wavelength band B3, and outputswaveform signals indicating the fluorescence intensities, respectively.

As shown in FIG. 10, on the basis of the waveform signals outputted bythe photodetectors, the analysis section 112 obtains, for example,graphs 51 and 52 in the case of a cell having a small expression amountof NF-κB, and obtains, for example, graphs 61 and 62 in the case of acell having a large expression amount of NF-κB. On the basis of awaveform signal outputted by a photodetector, the analysis section 112obtains a graph that corresponds to the nucleus. On the basis of thegraph regarding the nucleus obtained simultaneously with the graphs 51and 52, the analysis section 112 sets a width W1 of a waveform thatcorresponds to the nucleus in the graphs 51 and 52. On the basis of thegraph regarding the nucleus obtained simultaneously with the graphs 61and 62, the analysis section 112 sets a width W2 of a waveform thatcorresponds to the nucleus in the graphs 61 and 62.

According to the graph 51, the peak value of the fluorescence is betweenthresholds Sh1 and Sh2, and the peak of the waveform is present withinthe width W1 that corresponds to the nucleus. Thus, the analysis section112 can identify that NF-κB is localized in the nucleus. On the otherhand, according to the graph 52, the peak value of the fluorescence issmaller than the threshold Sh1. Thus, the analysis section 112 cannotidentify localization of NF-κB. According to the graph 61, the peakvalue of the fluorescence is greater than the threshold Sh2. Thus, theanalysis section 112 cannot identify localization of NF-κB. On the otherhand, according to the graph 62, the peak value of the fluorescence isbetween the thresholds Sh1 and Sh2, and a depression in the waveform ispresent within the width W2 that corresponds to the nucleus. Thus, theanalysis section 112 can identify that NF-κB is localized in thecytoplasm. Therefore, also in this case, as in the case where images areused as shown in FIG. 3, localization of NF-κB can be accuratelyidentified on the basis of two fluorescences having differentintensities.

Embodiment 2

In Embodiment 2, not by using two lights, but by using only the lighthaving the wavelength λ1, fluorescences having different intensitiesfrom each other are obtained. In Embodiment 2, among the steps of thecell information obtaining method shown in FIG. 1, only some proceduresin steps S1 and S2 are different from those in Embodiment 1. In thefollowing, the procedures that are different from those in Embodiment 1will be described.

In step S1, as shown in FIG. 11, NF-κB contained in each cell is labeledwith fluorescent substances 14 and 15 having different fluorescencewavelengths from each other. The fluorescent substances 14 and 15 areeach a fluorescent dye. When irradiated with light having the wavelengthλ1, the fluorescent substance 14 excites fluorescence having the samewavelength band as that of the fluorescent substance 11 in Embodiment 1.When irradiated with light having the wavelength λ1, the fluorescentsubstance 15 excites fluorescence having the same wavelength band asthat of the fluorescent substance 12 shown in FIG. 2. That is, for thefluorescent substances 14 and 15, the wavelength of the excitation lightis substantially the same.

In step S2, a sample containing the cells labeled with the fluorescentsubstances 14, 15, and 13 is caused to flow in the flow cell. Lightsrespectively having the wavelengths λ1 and λ3 are applied to each cellflowing in the flow cell, whereby fluorescences are generated from thefluorescent substances 14, 15, and 13, respectively. The fluorescencesgenerated from the fluorescent substances 14 and 15 are passed throughthe filter members 21 and 22, respectively, thereby becomingfluorescences having the wavelength bands B1 and B2, respectively. Here,the fluorescent substances 14 and 15 are configured such that thefluorescence having the wavelength band B1 has a high intensity and thefluorescence having the wavelength band B2 has a low intensity.

Compared with Embodiment 1, according to the apparatus configuration inEmbodiment 2, the light source 302, the condenser lens 312, and thedichroic mirror 321 are omitted in the optical detection unit 130 shownin FIG. 6.

Also in Embodiment 2, as in Embodiment 1, high intensity fluorescencehaving the wavelength band B1 and low intensity fluorescence having thewavelength band B2 are generated, whereby a high intensity fluorescenceimage and a low intensity fluorescence image can be obtained.Accordingly, as in Embodiment 1, on the basis of the high intensityfluorescence image and the low intensity fluorescence image, NF-κBhaving diverse distribution and amount in the cell can be accuratelyanalyzed.

Embodiment 3

In Embodiment 3, not by using two lights and two fluorescent substances,but by using a single light having the wavelength λ1 and a singlefluorescent substance 11, fluorescences having different intensitiesfrom each other are obtained. In Embodiment 3, among the steps of thecell information obtaining method shown in FIG. 1, only some proceduresin steps S1 and S2 are different from those in Embodiment 1. In thefollowing, the procedures that are different from those in Embodiment 1will be described.

In step S1, as shown in FIG. 12, NF-κB contained in each cell is labeledwith only the fluorescent substance 11 as used in Embodiment 1. At thistime, the fluorescent substance 11 may be bound to NF-κB via a singleantibody. In step S2, a sample containing the cells labeled with thefluorescent substances 11 and 13 is caused to flow in the flow cell.Lights respectively having the wavelengths λ1 and 23 are applied to eachcell flowing in the flow cell, whereby fluorescences are generated fromthe fluorescent substance 11 and 13, respectively.

As shown in FIG. 12, the fluorescence generated from the fluorescentsubstance 11 is divided into two, one of which is caused to pass throughthe filter member 21 as used in Embodiment 1, and the other of which iscaused to pass through the filter member 22 as used in Embodiment 1. Thefilter member 21 allows only light having the wavelength band B1 to passtherethrough. The filter member 22 allows only light having a wavelengthband B4 to pass therethrough. As shown in FIG. 13, the wavelength bandB1 includes, for example, a wavelength at which the intensity of thefluorescence generated from the fluorescent substance 11 peaks. Thewavelength band B4 is set to, for example, a wavelength band thatcontains longer wavelengths than those of the wavelength band B1, andthat does not overlap the wavelength band B1. As a result, as shown inFIG. 12, the fluorescence having the wavelength band B1 and havingpassed through the filter member 21 has a high intensity, and thefluorescence having the wavelength band B4 and having passed through thefilter member 22 has a low intensity.

It should be noted that the wavelength band B1 may not necessarilycontain a wavelength at which the intensity of the fluorescencegenerated from the fluorescent substance 11 peaks. The wavelength bandB4 may be set to a wavelength band that contains shorter wavelengthsthan those of the wavelength band B1. The wavelength band B4 maypartially overlap the wavelength band B1.

Examination of Embodiment 3

Next, examination of Embodiment 3 performed by the inventor will bedescribed.

1. Preparation

As the cells, human cardiac microvascular endothelial cell (HMVEC-C)(Lonza CatNo.CC-7030, Lot No.0000296500 (P4)) were obtained. As theprimary antibody, NF-κB p65 (D14E12) XP Rabbit mAb (Cell SignalingTechnologies #8242S) was obtained. As the secondary antibody, Goatanti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 647 conjugate(Life technologies A-21245) was obtained. To the secondary antibody,Alexa Fluor 647 was bound, as the fluorescent dye. Other than these,EGM-2MV Medium (Lonza Cat No.CC-3202), EGM-2MV SingleQuots Kit (LonzaCat No. CC-3202), PBS pH7.4 (GIBCO Cat No.10010-023), BSA (LAMPIRE CatNo. 7500805), PFA (WAKO Cat No.160-16061), and TritonX100 (NacalaiTesque CatNo.35501-15) were obtained.

2. Reagent Preparation

EGM-2MV SingleQuots Kit was added to 500 mL of EGM-2MV Medium, to createa culture medium. Paraformaldehyde was dissolved in pH12 PBS so as tohave a final concentration of 8% w/v, and then, the pH was adjusted to7.4. 1.5 g of BSA was added to and dissolved in PBS, and PBS wasadditionally added thereto to obtain 50 mL, whereby 3% BSA/PBS wasprepared. 0.5 g of BSA was added to and dissolved in PBS, and PBS wasadditionally added thereto to obtain 50 mL, whereby 1% BSA/PBS wasprepared. TritonX100 was adjusted with PBS so as to have a finalconcentration of 0.1% w/v.

3. Procedure

HMVEC-C cells were cultured in the EGM-2MV culture medium in accordancewith a manufacturer-recommended protocol. Cells within six passagesafter the purchase thereof were used in this examination. The shelf lifeof the culture medium after opening was set to three weeks. ForTNF-α-stimulated culture, the culture supernatant of about 70% confluentHMVEC-C cells was removed, and an EGM-2MV culture medium to whichRecombinant Human TNF-alpha had been added so as to have a finalconcentration of 25 ng/mL was added. Then, the resultant mixture wasleft still for 1 hour in a 37° C. CO² incubator. The culture medium wasremoved with an electric pipette, with about 3 mL left, and the cellswere detached with a scraper. 8% PFA/PBS was added by an amountequivalent to the collected suspension, and the resultant mixture wasallowed to react at room temperature for 15 minutes. At roomtemperature, centrifugal separation was performed at 1000 rpm for 3minutes. The cell pellet was washed with 1 mL of PBS twice. Thesupernatant was removed, and 1 mL of 0.1% Triton X-100/PBS was added.Then, the resultant mixture was allowed to react at room temperature for15 minutes. At room temperature, centrifugal separation was performed at1000 rpm for 3 minutes. The cells were washed with 1 mL of 1% BSA/PBStwice. The supernatant was removed, and 1 mL of 3% BSA/PBS was added.Then, the resultant mixture was left still for 30 minutes at roomtemperature. 400 μL of the primary antibody diluted at a ratio of 1/1600in 3% BSA/PBS was added. The resultant mixture was allowed to react atroom temperature for 1 hour. At room temperature, centrifugal separationwas performed at 1000 rpm for 3 minutes. The cells were washed with 1 mLof 1% BSA/PBS. 400 μL of the secondary antibody diluted at a ratio of1/1000 in 3% BSA/PBS was added. The resultant mixture was allowed toreact at room temperature for 30 minutes. The cells were washed with 1mL of 1% BSA/PBS twice. The supernatant was removed and 50 μL of 1%BSA/PBS was added.

4. Detection by Flow Cytometer

As a flow cytometer that can obtain fluorescence images, ImageStreamXMark II Imaging Flow Cytometer (Merck Millipore) was used. A sampleprepared in accordance with the procedure 3 above was caused to flow inthe flow cell of the flow cytometer. A laser light having a wavelengthof 647 nm was applied to the sample flowing in the flow cell. The laserlight having the wavelength of 647 nm corresponds to the laser lighthaving the wavelength λ1 shown in FIG. 12. The emission power of thelaser light having the wavelength of 647 nm was set to 10 mW. As aresult of the application of the laser light having the wavelength of647 nm to the fluorescent dye labeling NF-κB, fluorescence wasgenerated.

In the flow cytometer above, an image of the fluorescence generated dueto the laser light having the wavelength of 647 nm was taken via afilter member having a transmission wavelength band of 642 nm to 740 nm,whereby a high intensity fluorescence image was obtained. An image ofthe fluorescence generated due to the laser light having the wavelengthof 647 nm was taken via a filter member having a transmission wavelengthband of 740 nm to 800 nm, whereby a low intensity fluorescence image wasobtained. In the flow cytometer above, light having unnecessarywavelength bands is removed by a filter member or the like such thatlight having a target wavelength band is appropriately incident on thelight receiver.

With reference to FIGS. 14A and 14B, images obtained through thedetection above will be described.

“HIGH INTENSITY FLUORESCENCE” and “LOW INTENSITY FLUORESCENCE” are animage based on high intensity fluorescence generated from thefluorescent dye labeling NF-κB, and an image based on low intensityfluorescence generated from the fluorescent dye labeling NF-κB,respectively. In FIGS. 14A and 14B, the two images arranged along thehorizontal direction are images obtained from one cell. The respectiveimages are gray scale expressions of the obtained color images, made forconvenience. In each image, the white portion indicates that theintensity of the fluorescence is high.

In the case of the three cells shown in FIG. 14A, in the image based onthe low intensity fluorescence, the intensity is too low. Thus,localization of NF-κB is difficult to be identified. On the other hand,in the image based on the high intensity fluorescence, the intensity isappropriate. Thus, it is possible to identify that NF-κB is localized inthe cytoplasm. In the case of the three cells shown in FIG. 14B, in theimage based on the high intensity fluorescence, the intensity is toohigh. Thus, localization of NF-κB is difficult to be identified. On theother hand, in the image based on the low intensity fluorescence, theintensity is appropriate. Thus, it is possible to identify that NF-κB islocalized in the cytoplasm.

As described above, this examination shows that, when localization is tobe identified on the basis of two fluorescence images having differentintensities as in Embodiment 3, localization of NF-κB can be identified.Thus, according to Embodiment 3, if fluorescence generated from one kindof fluorescent dye is allowed to pass through the filter members 21 and22 which respectively allow fluorescences having different wavelengthbands to pass therethrough, and two fluorescence images are obtainedaccordingly, it is possible to accurately identify localization of NF-κBthrough a single measurement for one cell.

Apparatus Configuration in Embodiment 3

Compared with Embodiment 1, according to the apparatus configuration inEmbodiment 3 as shown in FIG. 15, the light source 302, the condenserlens 312, and the dichroic mirror 321 are omitted, and filter members414 and 425 are provided instead of the filter members 412 and 422, inthe optical detection unit 130 shown in FIG. 6. Among the lights thathave passed through the filter member 411, the filter member 414reflects the light having the wavelength band B4 and allows light otherthan the light having the wavelength band B4 to pass therethrough. Ofthe light reflected by the filter member 414, the filter member 425allows only the light having the wavelength band B4 to passtherethrough, and blocks light other than the light having thewavelength band B4. In this manner, the filter members 414 and 425 areeach configured to be able to separate only the fluorescence having thewavelength band B4, among the lights generated at the flow cell 200. Thelight receiver 502 takes an image of low intensity fluorescence havingthe wavelength band B4.

Also in Embodiment 3, as in Embodiment 1, high intensity fluorescenceand low intensity fluorescence are separately generated, whereby a highintensity fluorescence image and a low intensity fluorescence image canbe obtained. Accordingly, as in Embodiment 1, on the basis of the highintensity fluorescence image and the low intensity fluorescence image,NF-κB having diverse distribution and amount in the cell can beaccurately analyzed.

Embodiment 4

In Embodiment 4, among the steps of the cell information obtainingmethod shown in FIG. 1, only some procedures in steps S1 and S2 aredifferent from those in Embodiment 1. In the following, the proceduresdifferent from those in Embodiment 1 will be described.

In step S1, as shown in FIG. 16, NF-κB contained in each cell is labeledwith only the fluorescent substance 11 as used in Embodiment 1. In stepS2, a sample containing the cells labeled with the fluorescentsubstances 11 and 13 is caused to flow in the flow cell. Lightsrespectively having the wavelengths λ1 and λ3 are applied to each cellflowing in the flow cell, whereby fluorescences are generated from thefluorescent substances 11 and 13, respectively. At this time, the lighthaving the wavelength λ1 is applied at a high power to the cell, andthus, the fluorescence having the wavelength band B1 and having passedthrough the filter member 21 has a high intensity as in Embodiment 1.Further, this cell is moved in the flow cell, and light having thewavelength λ1 is applied to this moved cell, whereby fluorescence isgenerated from the fluorescent substance 11. At this time, the lighthaving the wavelength λ1 is applied at a low power to the cell.Accordingly, fluorescence having the wavelength band B1 having passedthrough the filter member 21 has a low intensity.

Compared with Embodiment 1, according to the apparatus configuration inEmbodiment 4, the light source 302, the condenser lens 312, the dichroicmirror 321, the filter members 412 and 422, the condenser lens 432, andthe light receiver 502 are omitted in the optical detection unit 130shown in FIG. 6. Further, compared with Embodiment 1, according to theapparatus configuration in Embodiment 4 as shown in FIG. 17, a lightsource 305 and a condenser lens 315 are added to the light applicationunit 300, and a condenser lens 471, a filter member 472, and a condenserlens 473 are added to the light condensing unit 400. Further, comparedwith Embodiment 1, according to the apparatus configuration inEmbodiment 4, a light receiver 505 is added.

FIG. 17 is a view at the time when the optical detection unit 130 isviewed in a direction parallel to the XY plane. For convenience, FIG. 17shows the light application unit 300 viewed in the Y-axis positivedirection, and the light condensing unit 400 and the light receiver 505viewed in the X-axis negative direction.

Light having the wavelength λ1 and emitted from the light source 301 isapplied to a position 211 in the flow pass 210 of the flow cell 200. Thelight source 305 has the same configuration as the light source 301, andemits light at a lower power than the light source 301. The condenserlens 315 condenses the light emitted from the light source 305, onto theposition 212 located to the Z-axis positive side relative to theposition 211 in the flow pass 210. The condenser lens 471 condensesfluorescence generated at the position 212. Of the light condensed bythe condenser lens 471, the filter member 472 allows only light havingthe wavelength band B1 to pass therethrough. The light receiver 505receives low intensity light having the wavelength band B1 and condensedby the condenser lens 473, and outputs, as an image pickup signal, imageinformation based on the received light.

A time period T in which a cell is moved from the position 211 to theposition 212 is obtained in advance. Thus, when the time period Telapses after a light based on a certain cell has been received by thelight receiver 501, the light based on the same cell is received by thelight receiver 505. Therefore, an image obtained on the basis of thelight receiver 501 can be associated with an image obtained on the basisof the light receiver 505, as images obtained from the same cell.

Also in Embodiment 4, as in Embodiment 1, high intensity fluorescenceand low intensity fluorescence are separately generated, whereby a highintensity fluorescence image and a low intensity fluorescence image canbe obtained. Accordingly, as in Embodiment 1, on the basis of the highintensity fluorescence image and the low intensity fluorescence image,NF-κB having diverse distribution and amount in the cell can beaccurately analyzed.

Embodiment 5

In Embodiment 5, among the steps of the cell information obtainingmethod shown in FIG. 1, only some procedures in step S2 are differentfrom those in Embodiment 1. In the following, the procedures differentfrom those in Embodiment 1 will be described.

In step S2, as shown in FIG. 18, a sample containing the cells labeledwith the fluorescent substances 11 to 13 is caused to flow in the flowcell. Lights respectively having the wavelengths λ1 to λ3 are applied toeach cell flowing in the flow cell, whereby fluorescences are generatedfrom the fluorescent substances 11 to 13, respectively. Thefluorescences generated from the fluorescent substances 11 and 12 arecombined to be incident onto a filter member 24. The filter member 24 isimplemented by a prism. Due to the difference in the wavelength band,the combined fluorescence generated from the fluorescent substances 11and 12 is split by the filter member 24 into a fluorescence having thewavelength band B1 and a fluorescence having the wavelength band B2.Here, as in Embodiment 1, the light having the wavelength λ1 is appliedat a high power to the cell, and the light having the wavelength λ2 isapplied at a low power to the cell. As a result, as in Embodiment 1, thefluorescence having the wavelength band B1 and having passed through thefilter member 21 has a high intensity, and the fluorescence having thewavelength band B2 and having passed through the filter member 22 has alow intensity.

It should be noted that fluorescences generated from the fluorescentsubstances 11 to 13 may be combined to be incident onto the filtermember 24, and then, due to the difference in the wavelength band, thecombined fluorescence may be split by the filter member 24 intofluorescences having the wavelength bands B1 to B3, respectively. In theexample shown here, the filter member 24 is used in the configuration ofEmbodiment 1, but the filter member 24 may be used in the configurationsof Embodiments 2 to 4.

Compared with Embodiment 1, according to the apparatus configuration inEmbodiment 5 as shown in FIG. 19, in the optical detection unit 130shown in FIG. 6, the filter members 411, 412, 421, and 422 are omitted,and a filter member 481 is added to the light condensing unit 400. Thefilter member 481 is implemented by a prism.

Fluorescences generated from a sample flowing in the flow cell 200 areincident on the filter member 481. The fluorescences incident on thefilter member 481 go out from the filter member 481 at different anglesaccording to their fluorescence wavelengths, respectively. The condenserlens 431 and the light receiver 501 are disposed in a direction thatcorresponds to the fluorescence having the wavelength band B1, of thefluorescences going out from the filter member 481. Accordingly, thelight receiver 501 can take an image of high intensity fluorescencehaving the wavelength band B1. The condenser lens 432 and the lightreceiver 502 are disposed in a direction that corresponds to thefluorescence having the wavelength band B2, of the fluorescences goingout from the filter member 481. Accordingly, the light receiver 502 cantake an image of low intensity fluorescence having the wavelength bandB2.

Also in Embodiment 5, as in Embodiment 1, high intensity fluorescenceand low intensity fluorescence are separately generated, whereby a highintensity fluorescence image and a low intensity fluorescence image canbe obtained. Accordingly, as in Embodiment 1, on the basis of the highintensity fluorescence image and the low intensity fluorescence image,NF-κB having diverse distribution and amount in the cell can beaccurately analyzed.

Embodiment 6

In Embodiment 6, a test substance contained in a cell is brought intocontact with a substrate, thereby causing a fluorescent substance to begenerated; then, light is applied to the generated fluorescentsubstance; and then, on the basis of fluorescence that has beengenerated from the fluorescent substance upon the application of thelight, the localization state of the test substance is identified. InEmbodiment 6, the test substance is cytoplasm. The substrate includes acleavage site at which the substrate is cleaved upon contact with thetest substance, and when the substrate is cleaved at the cleavage site,the substrate generates a fluorescent substance. More specifically, whenthe substrate comes into contact with the cytoplasm being the testsubstance, the substrate is cleaved by an enzyme present in thecytoplasm. In Embodiment 6, localization of the cytoplasm is judged onthe basis of fluorescence from the fluorescent substance labeling thecytoplasm. It should be noted that the test substance may be, forexample, a substance other than cytoplasm contained in a cell, such asprotein in cytoplasm, organelle, cell membrane, or the like.

In Embodiment 6, among the steps of the cell information obtainingmethod shown in FIG. 1, some procedures in step S1 are different fromthose in Embodiment 3. In the following, the procedures different fromthose in Embodiment 3 will be described.

In step S1, as shown in FIG. 20, the cell and a substrate 16 a are mixedtogether. The substrate 16 a generates a fluorescent substance 16 b whenbeing hydrolyzed with esterase contained in the cytoplasm. When the celland the substrate 16 a are mixed together, the substrate 16 a havingpassed through the cell membrane comes into contact with the cytoplasm,thereby being hydrolyzed with the esterase contained in the cytoplasm,to generate the fluorescent substance 16 b. Accordingly, the cytoplasmis labeled with the fluorescent substance 16 b.

Subsequently, the processes of step S2 and thereafter will be performedas in Embodiment 3. That is, in step S2, a sample containing the cellslabeled with the fluorescent substance 16 b is caused to flow in theflow cell. Then, as shown in FIG. 20, lights respectively having thewavelengths λ1 and λ3 are applied to each cell flowing in the flow cell,whereby fluorescences are generated from the fluorescent substances 16 band 13, respectively. Then, as shown in FIG. 20, the fluorescencegenerated from the fluorescent substance 16 b is divided into two, oneof which is caused to pass through the filter member 21, and the otherof which is caused to pass through the filter member 22. As a result, asin Embodiment 3, the fluorescence having the wavelength band B1 andhaving passed through the filter member 21 has a high intensity, and thefluorescence having the wavelength band B4 and having passed through thefilter member 22 has a low intensity. It should be noted that theapparatus according to Embodiment 6 has the same configuration as thatin Embodiment 3.

Also in Embodiment 6, as in Embodiment 3, two fluorescences respectivelyhaving different intensities are generated, whereby a high intensityfluorescence image and a low intensity fluorescence image can beobtained. Accordingly, on the basis of the high intensity fluorescenceimage and the low intensity fluorescence image, localization of thecytoplasm can be accurately identified. Thus, if localization of thecytoplasm can be accurately identified, the range of the cytoplasm canbe precisely defined. Accordingly, for example, if the range of thecytoplasm is combined with another analysis, further detailed analysisis enabled. Moreover, for example, the range of the cytoplasm can beutilized in study of cells and the like.

Examination of Embodiment 6

Next, examination of Embodiment 6 performed by the inventor will bedescribed.

1. Preparation

As the cells, human cardiac microvascular endothelial cells (HMVEC-C)(Lonza CatNo.CC-7030, Lot No.0000296500 (P4)) were obtained. As thecytoplasm labeling reagent, Cell Explorer Fixable Live Cell Tracking Kit*Green Fluorescence* (Cosmo Bio 22621) was obtained. The cytoplasmlabeling reagent contains a substance that corresponds to the substrate16 a shown in FIG. 20. The cytoplasm labeling reagent is hydrophobic. Bypassing through the cell membrane and being hydrolyzed with esterase inthe cell, the cytoplasm labeling reagent generates a fluorescentsubstance. The fluorescent substance generated here corresponds to thefluorescent substance 16 b shown in FIG. 20. As the nucleus stainingdye, Cellstain Hoechst 33342 solution (DOjinDO 11342) was obtained.Other than these, EGM-2MV Medium (Lonza Cat No.CC-3202), EGM-2MVSingleQuots Kit (Lonza Cat No. CC-3202), PBS pH7.4 (GIBCO CatNo.10010-023), BSA (LAMPIRE Cat No. 7500805), PFA (WAKO CatNo.160-16061), and TritonX100 (Nacalai Tesque CatNo.35501-15) wereobtained.

2. Reagent Preparation

EGM-2MV SingleQuots Kit was added to 500 mL of EGM-2MV Medium, to createa culture medium. Paraformaldehyde was dissolved in pH12 PBS so as tohave a final concentration of 8% w/v, and then the pH was adjusted to7.4. 1.5 g of BSA was added to and dissolved in PBS, and PBS wasadditionally added thereto to obtain 50 mL, whereby 3% BSA/PBS wasprepared. 0.5 g of BSA was added to and dissolved in PBS, and PBS wasadditionally added thereto to obtain 50 mL, whereby 1% BSA/PBS wasprepared. TritonX100 was adjusted with PBS to have a final concentrationof 0.1% w/v. 100 μL of DMSO was added to a Track kit Green vial tocreate a 1000×Track kit Green stock solution, and by adding a 1/1000amount thereof to an Assay buffer of the Kit, a Track kit workingsolution was prepared.

3. Procedure

HMVEC-C cells were cultured in the EGM-2MV culture medium in accordancewith a manufacturer-recommended protocol. Cells within six passagesafter the purchase thereof were used in this examination. The shelf lifeof the culture medium was set to three weeks. After the cell culturebecame 70% confluent, the culture medium was removed with an electricpipette, with about 3 mL of the culture medium left, and the cells weredetached with a scraper. 3 μL of the Track kit working solution wasadded to the collected 3 mL suspension, and the resultant mixture wasleft still for 30 minutes in an 37° C. CO² incubator. After the mixturewas left still for 30 minutes, the mixture was centrifuged at 1000 rpmfor 3 minutes at room temperature. The cell pellet was washed with 5 mLof PBS three times. The supernatant was removed and 50 μL of 1% BSA/PBSwas added.

4. Detection by Flow Cytometer

As a flow cytometer that can obtain fluorescence images, ImageStreamXMark II Imaging Flow Cytometer (Merck Millipore) was used. A sampleprepared in accordance with the procedure 3 above was caused to flow inthe flow cell of the flow cytometer. Laser lights respectively havingwavelengths of 488 nm and 405 nm were applied to the sample flowing inthe flow cell. The laser lights having the wavelengths of 488 nm and 405nm correspond to the laser lights having the wavelengths λ1 and λ3 shownin FIG. 20, respectively. The emission powers of the laser lights havingthe wavelengths of 488 nm and 405 nm were set to 50 mW and 20 mW,respectively. As a result of the application of the laser light havingthe wavelength of 488 nm to the fluorescent dye labeling the cytoplasm,fluorescence was generated. As a result of the application of the laserlight having the wavelength of 405 nm to the nucleus staining dye,fluorescence was generated.

In the flow cytometer above, an image of the fluorescence generated dueto the laser light having the wavelength of 488 nm was taken via afilter member having a transmission wavelength band of 480 nm to 560 nm,whereby a high intensity fluorescence image was obtained. An image ofthe fluorescence generated due to the laser light having the wavelengthof 488 nm was taken via a filter member having a transmission wavelengthband of 560 nm to 595 nm, whereby a low intensity fluorescence image wasobtained. An image of the fluorescence generated due to the laser lighthaving the wavelength of 405 nm was taken via a filter member having atransmission wavelength band of 420 nm to 505 nm, and a fluorescenceimage corresponding to the nucleus was obtained. Furthermore, a laserlight whose wavelength was set between 420 nm to 480 nm was applied tothe sample flowing in the flow cell. An image of the light obtained as aresult of this laser light having passed through the cell was taken viathe filter member having a transmission wavelength band of 420 nm to 480nm, whereby a bright field image was obtained. In the flow cytometerabove, light having unnecessary wavelength bands is removed by a filtermember or the like such that light having a target wavelength band isappropriately incident on the light receiver.

With reference to FIG. 21, images obtained through the detection abovewill be described.

“BRIGHT FIELD” indicates a bright field image of a cell. “HIGH INTENSITYFLUORESCENCE” and “LOW INTENSITY FLUORESCENCE” respectively correspondto an image based on high intensity fluorescence generated from thefluorescent dye labeling the cytoplasm, and an image based on lowintensity fluorescence generated from the fluorescent dye labeling thecytoplasm. “FLUORESCENCE FROM NUCLEUS” corresponds to an image based onfluorescence generated from the nucleus staining dye that stains thenucleus. The four images arranged along the horizontal direction areimages obtained from one cell. The images other than the bright fieldimage are gray scale expressions of the obtained color images, made forconvenience. In the images other than the bright field image, the whiteportion indicates that the intensity of the fluorescence is high.

In the case of the cell at the top row and the cell in the second rowfrom the top, in the image based on the low intensity fluorescence, theintensity is too low. Thus, localization of the cytoplasm is not clear.On the other hand, in the case of the image based on the high intensityfluorescence, the intensity is appropriate. Thus, it is possible toaccurately identify localization of the cytoplasm. In the case of thecell in the lowest row and the cell in the second lowest row, in theimage based on the high intensity fluorescence, the intensity is toohigh. Thus, localization of the cytoplasm is difficult to be identified.On the other hand, in the case of the image based on the low intensityfluorescence, the intensity is appropriate. Thus, localization of thecytoplasm can be accurately identified.

As described above, this examination shows that localization of thecytoplasm can be identified on the basis of the two fluorescence imagesrespectively having different intensities as in Embodiment 6. Thus,according to Embodiment 6, if fluorescence generated from one kind offluorescent dye labeling the cytoplasm is allowed to pass through thefilter members 21 and 22 which respectively allow fluorescences havingdifferent wavelength bands to pass therethrough, and two fluorescenceimages can be obtained, accordingly, it is possible to accuratelyidentify localization of the cytoplasm through a single measurement forone cell.

Embodiment 7

In Embodiment 7, a test substance contained in a cell is brought intocontact with two kinds of substrates, thereby causing two kinds offluorescent substances to be generated; then light is applied to thegenerated two kinds of fluorescent substances; and then, on the basis ofthe fluorescences that have been generated from the two kinds offluorescent substances upon the application of the light, thelocalization state of the test substance is identified. That is, inEmbodiment 7, not by using one light as in Embodiment 6, but by usinglights respectively having different wavelengths, fluorescencesrespectively having different intensities are obtained. It should benoted that two kinds of fluorescent substances may be generated bybringing the test substance into contact with one kind of substrate.

In Embodiment 7, among the step of the cell information obtaining methodshown in FIG. 1, only some procedures in steps S1 and S2 are differentfrom those in Embodiment 6. In the following, the procedures differentfrom those in Embodiment 6 will be described.

In step S1, as shown in FIG. 22, the cell and substrates 17 a and 18 aare mixed together. The substrates 17 a and 18 a generate fluorescentsubstances 17 b and 18 b respectively when being hydrolyzed withesterase contained in the cytoplasm. The fluorescent substances 17 b and18 b are configured to excite fluorescences having different wavelengthbands from each other when being irradiated with lights having thewavelengths λ1 and λ2, respectively. When the cell and the substrates 17a and 18 a are mixed together, the substrates 17 a and 18 a havingpassed through the cell membrane come into contact with the cytoplasm,thereby being hydrolyzed with esterase contained in the cytoplasm, togenerate the fluorescent substances 17 b and 18 b. Accordingly, thecytoplasm is labeled with the fluorescent substances 17 b and 18 b.

In step S2, a sample containing the cells labeled with the fluorescentsubstances 17 b and 18 b are caused to flow in the flow cell. Then, asshown in FIG. 22, lights respectively having the wavelengths λ1, λ2, andλ3 are applied to each cell flowing in the flow cell, wherebyfluorescences are generated from the fluorescent substances 17 b, 18 b,and 13, respectively. At this time, the laser light having thewavelength λ1 is applied to the cell at a high power, and the laserlight having the wavelength λ2 is applied to the cell at a low power.The fluorescence generated from the fluorescent substance 17 b is passedthrough the filter member 21, thereby becoming fluorescence having thewavelength band B1. The fluorescence generated from the fluorescentsubstance 18 b is passed through the filter member 22, thereby becomingfluorescence having the wavelength band B2. As a result, thefluorescence having the wavelength band B1 and having passed through thefilter member 21 has a high intensity, and the fluorescence having thewavelength band B2 and having passed through the filter member 22 has alow intensity. It should be noted that the apparatus according toEmbodiment 7 has the same configuration as that in Embodiment 1.

Also in Embodiment 7, as in Embodiment 6, two fluorescences respectivelyhaving different intensities are generated, whereby a high intensityfluorescence image and a low intensity fluorescence image can beobtained. Accordingly, as in Embodiment 6, on the basis of the highintensity fluorescence image and the low intensity fluorescence image,localization of the cytoplasm can be accurately identified.

What is claimed is:
 1. A cell information obtaining method comprising:causing a plurality of fluorescent substances having differentfluorescence wavelengths from each other to be bound to a test substancecontained in a cell; applying light to the cell to cause fluorescenceshaving different wavelengths and intensities to be generated from theplurality of fluorescent substances; and obtaining a first fluorescenceinformation and a second fluorescence information on the basis of thegenerated fluorescences.
 2. The cell information obtaining method ofclaim 1, wherein a distribution state of the test substance in the cellis identified on the basis of the first fluorescence information and thesecond fluorescence information.
 3. The cell information obtainingmethod of claim 1, wherein a localization state of the test substance inthe cell is identified on the basis of the first fluorescenceinformation and the second fluorescence information.
 4. The cellinformation obtaining method of claim 1, wherein whether the testsubstance is nuclearly localized or cytoplasmically localized isidentified on the basis of the first fluorescence information and thesecond fluorescence information.
 5. The cell information obtainingmethod of claim 1, wherein wavelengths for excitation light for theplurality of fluorescent substances are different from each other. 6.The cell information obtaining method of claim 1, wherein first lightand second light which has a lower intensity than that of the firstlight are applied to the cell.
 7. The cell information obtaining methodof claim 1, wherein wavelengths and intensities of fluorescencesgenerated from the plurality of fluorescent substances by beingirradiated with lights having the same wavelength are different fromeach other.
 8. The cell information obtaining method of claim 1, whereinthe fluorescences generated from the plurality of fluorescent substancesare separated into a first fluorescence and a second fluorescence whichhas a lower intensity than that of the first fluorescence.
 9. A cellinformation obtaining method comprising: causing a fluorescent substanceto be bound to a test substance contained in a cell; applying light tothe cell to cause a fluorescence to be generated from the fluorescentsubstance; obtaining, from the generated fluorescence, a plurality offluorescences having different wavelengths and intensities; obtaining afirst fluorescence information and a second fluorescence information onthe basis of the obtained fluorescences; and identifying a distributionstate of the test substance in the cell on the basis of the firstfluorescence information and the second fluorescence information. 10.The cell information obtaining method of claim 9, wherein a localizationstate of the test substance in the cell is identified on the basis ofthe plurality of pieces of fluorescence information.
 11. The cellinformation obtaining method of claim 9, wherein the fluorescencesgenerated from the fluorescent substance are separated into a firstfluorescence and a second fluorescence which has a lower intensity thanthat of the first fluorescence.
 12. The cell information obtainingmethod of claim 1, further comprising: obtaining a first fluorescenceimage of a fluorescence having a first wavelength and having beengenerated from the cell irradiated with first light, and obtaining asecond fluorescence image of a fluorescence having a second wavelengthand having been generated from the cell irradiated with second lightwhich has a lower intensity than that of the first light.
 13. The cellinformation obtaining method of claim 1, wherein a sample containing thecell is caused to flow in a flow cell, and light is applied to the cellflowing in the flow cell, to cause the fluorescence to be generated. 14.The cell information obtaining method of claim 3, wherein theidentification of the localization state of the test substance includescalculation of a proportion of a localization amount of the testsubstance in an analysis target site in the cell relative to alocalization amount of the test substance in an entirety of the cell.15. The cell information obtaining method of claim 3, wherein theidentification of the localization state of the test substance isperformed on the basis of, among the first fluorescence information andthe second fluorescence information obtained from the plurality offluorescences having different intensities, fluorescence informationobtained from a fluorescence whose intensity is included in apredetermined range.
 16. The cell information obtaining method of claim3, wherein the identification of the localization state of the testsubstance is performed on the basis of, among the first fluorescenceinformation and the second fluorescence information obtained from theplurality of fluorescences having different intensities, fluorescenceinformation in which difference between the intensity of thefluorescence at an analysis target site in the cell and the intensity ofthe fluorescence in a portion, other than the analysis target site, ofthe cell is greater than a predetermined threshold.
 17. The cellinformation obtaining method of claim 1, wherein the test substance is aprotein, mRNA, microRNA, cytoplasm, organelle, or cell membrane.
 18. Thecell information obtaining method of claim 1, wherein a proportion orthe number of cells in which the test substance is localized in aspecific site among the cells contained in the sample is calculated onthe basis of the first fluorescence information and the secondfluorescence information.
 19. A cell information obtaining apparatuscomprising: a light application unit configured to apply light to a cellcontaining a test substance to which a plurality of fluorescentsubstances having different fluorescence wavelengths from each other arebound, thereby to cause fluorescences having different wavelengths andintensities to be generated from the plurality of fluorescentsubstances; a light receiver configured to receive the fluorescencesgenerated from the plurality of fluorescent substances; and an obtainingsection configured to obtain a first fluorescence information and asecond fluorescence information on the basis of the fluorescences havingdifferent intensities.
 20. The cell information obtaining apparatus ofclaim 19, wherein the light application unit includes: a first lightsource configured to apply first light; and a second light sourceconfigured to apply second light having a wavelength different from thatof the first light and having a lower intensity than that of the firstlight.